U.S. patent application number 11/598414 was filed with the patent office on 2007-03-08 for proteinase inhibitor, precursor thereof and genetic sequences encoding same.
Invention is credited to Marilyn Anne Anderson, Angela Hilary Atkinson, Adrienne Elizabeth Clarke, Robyn Louise Heath.
Application Number | 20070054365 11/598414 |
Document ID | / |
Family ID | 3776602 |
Filed Date | 2007-03-08 |
United States Patent
Application |
20070054365 |
Kind Code |
A1 |
Anderson; Marilyn Anne ; et
al. |
March 8, 2007 |
Proteinase inhibitor, precursor thereof and genetic sequences
encoding same
Abstract
The present invention relates generally to proteinase
inhibitors, a precursor thereof and to genetic sequences encoding
same. More particularly, the present invention relates to a nucleic
acid molecule comprising a sequence of nucleotides which encodes or
is complementary to a sequence which encodes a type II serine
proteinase inhibitor (PI) precursor from a plant wherein said
precursor comprises at least three PI monomers and wherein at least
one of said monomers has a chymotrypsin specific site and at least
one other of said monomers has a trypsin specific site.
Inventors: |
Anderson; Marilyn Anne;
(Keilor, AU) ; Atkinson; Angela Hilary; (Montrose,
AU) ; Heath; Robyn Louise; (Williamstown, AU)
; Clarke; Adrienne Elizabeth; (Parkville, AU) |
Correspondence
Address: |
SCULLY SCOTT MURPHY & PRESSER, PC
400 GARDEN CITY PLAZA
SUITE 300
GARDEN CITY
NY
11530
US
|
Family ID: |
3776602 |
Appl. No.: |
11/598414 |
Filed: |
November 13, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11178737 |
Jul 11, 2005 |
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11598414 |
Nov 13, 2006 |
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10157622 |
May 29, 2002 |
6946278 |
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11178737 |
Jul 11, 2005 |
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09431499 |
Nov 1, 1999 |
6451573 |
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10157622 |
May 29, 2002 |
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08454295 |
Sep 1, 1995 |
6031087 |
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PCT/AU93/00659 |
Dec 16, 1993 |
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09431499 |
Nov 1, 1999 |
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Current U.S.
Class: |
435/69.2 ;
435/184; 435/320.1; 435/325; 536/23.2 |
Current CPC
Class: |
C12N 2799/06 20130101;
C07K 14/811 20130101; G01N 2333/81 20130101; G01N 2500/02 20130101;
C12N 2799/026 20130101 |
Class at
Publication: |
435/069.2 ;
435/184; 435/320.1; 435/325; 536/023.2 |
International
Class: |
C12N 9/99 20060101
C12N009/99; C07H 21/04 20060101 C07H021/04; C12N 15/09 20060101
C12N015/09 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 16, 1992 |
AU |
PL 6399/92 |
Claims
1-28. (canceled)
29. A method of increasing or enhancing resistance of a plant to
insect or other pathogen infestation, said method comprising
introducing nucleic acid molecules to a cell or group of cells of a
plant wherein one nucleic acid molecule comprises a sequence of
nucleotides which encodes or is complementary to a sequence which
encodes a type II serine proteinase inhibitor (PI) or pre-cursor
thereof wherein said isolated nucleic acid has the nucleotide
sequence set forth in SEQ ID NO:1 or hybridizes to the nucleotide
sequence set forth in SEQ ID NO:1 under the conditions of at least
one of 4.times.SSC at room temperature, 2.times.SSC at room
temperature, 1.times.SSC at 40.degree. C., 2.times.SSC with 0.1%
w/v SDS at 68.degree. C. or 2.times.SSC with 1% w/v SDS at
68.degree. C., wherein said precursor comprises at least three PI
monomers and wherein at least one of said monomers has a
chymotrypsin specific site and at least one of said monomers has a
trypsin specific site and wherein another nucleic acid molecule
encodes an insecticide said method further comprising regenerating
a plant from the cells and growing said plant or progeny of said
plant for a time and under conditions sufficient to permit
expression of said nucleic acid molecules to produce a PI or
precursor thereof and an insecticide which inhibits growth and
infestation of said insect or pathogen.
30. The method according to claim 1 wherein said PI precursor
comprises at least four monomers.
31. The method according to claim 1 wherein the PI precursor
comprises at least five monomers.
32. The method according to claim 1 wherein the PI precursor
comprises at least six monomers.
33. The method according to claim 1 wherein one of the nucleic acid
molecules encodes a peptide selected from the group consisting of
SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8,
SEQ ID NO:9 and SEQ ID NO:10.
34. The method according to anyone of claims 29-33 wherein the
insecticide is Bt.
35. The method according to anyone of claims 29-33 wherein the
insecticide is a PI.
36. A transgenic plant carrying nucleic acid molecules wherein one
nucleic acid molecule comprises a sequence of nucleotides which
encodes or is complementary to a sequence which encodes a type II
serine proteinase inhibitor (PI) or precursor thereof which nucleic
acid molecule comprises the nucleotide sequence set forth in SEQ ID
NO: 1 or hybridizes to SEQ ID NO: 1 under the conditions of at
least one of 4.times.SSC at room temperature, 2.times.SSC at room
temperature, 1.times.SSC at 40.degree. C., 2.times.SSC with 0.1%
w/v SDS at 68.degree. C. or 2.times.SSC with 1% w/v SDS at
68.degree. C., wherein said precursor comprises at least three PI
monomers and wherein at least one of said monomers has a
chymotrypsin specific site and at least one of said monomers has a
trypsin specific site and the other nucleic acid molecule encodes
an insecticide.
37. The transgenic plant according to claim 36 wherein said
transgenic plant produces one or more PI monomers selected from the
list consisting of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID
NO:8 and SEQ ID NO:9.
38. The transgenic plant according to claim 36 wherein said
transgenic plant produces a peptide consisting of SEQ ID NO:4 or
SEQ ID NO:10.
39. The transgenic plant according to anyone of claims 36-38
wherein the insecticide is Bt.
40. The transgenic plant according to anyone of claims 36-38
wherein the insecticide is another PI.
41. Progeny or reproductive material of the transgenic plant
according to anyone of claims 36-40.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a continuation of Ser. No.
11/178,737, filed Jul. 11, 2005 which is a continuation of Ser. No.
10/157,622, filed May 29, 2002, now U.S. Pat. No. 6,946,278, which
is a continuation of Ser. No. 09/431,499, filed Nov. 1, 1999, now
U.S. Pat. No. 6,451,573, which is a divisional of Ser. No.
08/454,295, filed Sep. 1, 1995, now U.S. Pat. No. 6,031,087, which
corresponds to PCT/AU93/00659 having an international filing date
of Dec. 16, 1993.
[0002] The present invention relates generally to proteinase
inhibitors, a precursor thereof and to genetic sequences encoding
same.
[0003] Nucleotide and amino acid sequences are referred to herein
by sequence identity numbers (SEQ ID NOs) which are defined after
the bibliography. A general summary of the SEQ ID NOs is provided
before the examples.
[0004] Throughout this specification and the claims which follow,
unless the context requires otherwise, the word "comprise", or
variations such as "comprises" or "comprising", will be understood
to imply the inclusion of a stated element or integer or group of
elements or integers but not the exclusion of any other element or
integer or group of elements or integers.
[0005] Several members of the families Solanaceae and Fabaceae
accumulate serine proteinase inhibitors in their storage organs and
inleaves in response to wounding (Brown and Ryan, 1984, 1984;
Richardson, 1977). The inhibitory activities of these proteins are
directed against a wide range of proteinases of microbial and
animal origin, but rarely against plant proteinases (Richardson,
1977). It is believed that these inhibitors are involved in
protection of the plants against pathogens and predators. In potato
tubers and legume seeds, the inhibitors can comprise 10% or more of
the stored proteins (Richardson, 1977), while in leaves of tomato
and potato (Green and Ryan, 1972), and alfalfa (Brown & Ryan,
1984; Graham et al., 1986). High levels of these inhibitors (up to
50% of total soluble protein) are also present in unripe fruits of
the wild tomato, Lycopersicon peruvianum (Pearce et al., 1988).
[0006] There are two families of serine proteinase inhibitors in
tomato and potato (Ryan, 1984). Type I inhibitors are small
proteins (monomer Mr 8100) which inhibit chymotrypsin at a single
reactive site (Melville and Ryan, 1970; Plunkett et al., 1982).
Inhibitors of the type II family generally contain two reactive
sites, one of which inhibits chymotrypsin and the other trypsin
(Bryant et al., 1976; Plunkett et al., 1982). The type II
inhibitors have a monomer Mr of 12,300 (Plunkett et al., 1982).
Proteinase inhibitor I accumulates in etiolated tobacco (Nicotiana
tabacum) leaves (Kuo et al, 1984), and elicitors from Phytophthora
parasitica var. nicotianae were found to induce proteinase
inhibitor I accumulation in tobacco cell suspension cultures
(Rickauer et al, 1989).
[0007] There is a need to identify other proteinase inhibitors and
to investigate their potential use in the development of transgenic
plants with enhanced protection against pathogens and predators. In
accordance with the present invention, genetic sequences encoding a
proteinase inhibitor precursor have been cloned. The precursor has
multi-proteinase inhibitor domains and will be useful in developing
a range of transgenic plants with enhanced proteinase inhibitor
expression. Such plants will have enhanced protective properties
against pathogens and predators. The genetic constructs of the
present invention will also be useful in developing vaccines for
ingestion by insects which are themselves predators or which act as
hosts for plant pathogens. The recombinant precursor or monomeric
inhibitors will also be useful in topical sprays and in assisting
animals in feed digestion.
[0008] Accordingly, one aspect of the present invention relates to
a nucleic acid molecule comprising a sequence of nucleotides which
encodes or is complementary to a sequence which encodes a type II
serine proteinase inhibitor (PI) precursor from a plant wherein
said precursor comprises at least three PI monomers and wherein at
least one of said monomers has a chymotrypsin specific-site and at
least one other of said monomers has a trypsin specific site.
[0009] The "nucleic acid molecule" of the present invention may be
RNA or DNA (eg cDNA), single or double stranded and linear or
covalently closed. The nucleic acid molecule may also be genomic
DNA corresponding to the entire gene or a substantial portion
thereof or to fragments or derivatives thereof. The nucleotide
sequence may correspond to the naturally occurring nucleotide
sequence of the genomic or cDNA clone or may contain single or
multiple nucleotide substitutions, deletions and/or additions
thereto. All such variants in the nucleic acid molecule either
retain the ability to encode at least one monomer or active part
thereof or are useful as hybridisation probes or polymerase chain
reaction (PCR) primers for the same or similar genetic sequences in
other sources.
[0010] Preferably, the PI precursor comprises at least four, more
preferably at least five and even more preferably at least six PI
monomers. Still more preferably, the PI precursor further comprises
a signal sequence. The PI precursor of the present invention
preferably comprises amino acid sequences which are process sites
for cleavage into individual monomers.
[0011] The term "precursor" as used herein is not intended to place
any limitation on the utility of the precursor molecule itself or a
requirement that the molecule first be processed into monomers
before PI activity is expressed. The precursor molecule has PI
activity and the present invention is directed to the precursor and
to the individual monomers of the precursor.
[0012] Furthermore, the present invention extends to a nucleic acid
molecule comprising a sequence of nucleotides which encodes or is
complementary to a sequence which encodes a hybrid type II serine
PI precursor wherein said precursor comprises at least two monomers
from different PIs. The at least two monomers may be modified such
as being unable to be processed into individual monomers or may
retain the ability to be so processed. Preferably, at least one of
said monomers has a chymotrypsin specific site and the other of
said monomers has a trypsin specific site. Preferably there are at
least three monomers, more preferably at least four monomers, still
more preferably at least five monomers and yet still more
preferably at least six monomers wherein at least two are from
different PIs. In a most preferred embodiment, at least one of said
monomers is a thionin. Such hybrid PI precursors and/or monomers
thereof are particularly useful in generating molecules which are
"multi-valent" in that they are active against a range of pathogens
and predators such as both fungi and insects. Accordingly,
reference herein to "PI precursor" includes reference to hybrid
molecules.
[0013] The present invention is exemplified by the isolation of the
subject nucleic acid molecule from Nicotiana alata which has the
following nucleotide sequence (SEQ ID NO. 1) and a corresponding
amino acid sequence (SEQ ID NO. 3): TABLE-US-00001 AAG GCT TGT ACC
TTA AAC Lys Ala Cys Thr Leu Asn TGT GAT CCA AGA ATT GCC TAT GGA GTT
TGC CCG CGT TCA GAA GAA AAG Cys Asp Pro Arg Ile Ala Tyr Gly Val Cys
Pro Arg Ser Glu Glu Lys AAG AAT GAT CGG ATA TGC ACC AAC TGT TGG GCA
GGC ACG AAG GGT TGT Lys Asn Asp Arg Ile Cys Thr Asn Gys Gys Ala Gly
Thr Lys Gly Cys AAG TAC TTC AGT GAT GAT GGA ACT TTT GTT TGT GAA GGA
GAG TCT GAT Lys Tyr Phe Ser Asp Asp Gly Thr Phe Val Cys Glu Gly Glu
Ser Asp CCT AGA AAT CCA AAG GCT TGT ACC TTA AAC TGT GAT CCA AGA ATT
GCC Pro ArG Asn Pro Lys Ala Gys Thr Leu Asn Gys Asp Pro Arg Ile Ala
TAT GGA GTT TGC CCG CGT TCA GAA GAA AAG AAG AAT GAT CGG ATA TGC Tyr
Gly Val Cys Pro Arg Ser Glu Glu Lys Lys Asn Asp Arg Ile Cys ACC AAC
TGT TGC GCA GGC ACG AAG GGT TGT AAG TAC TTC AGT GAT GAT Thr Asn Cys
Cys Ala Gly Thr Lys Gly Gys Lys Tyr Phe Ser Asp Asp GGA ACT TTT GTT
TGT GAA GGA GAG TCT GAT CCT AGA AAT CCA AAG GCT Gly Thr Phe Val Cys
Glu Gly Glu Ser Asp Pro Arg Asn Pro Lys Ala TGT CCT CGG AAT TGC GAT
CCA AGA ATT GCC TAT GGG ATT TGC CCA CTT Cys Pro Arg Asn Cys Asp Pro
Arg Ile Ala Tyr Gly Ile Cys Pro Leu GCA GAA GAA AAG AAG AAT GAT CGG
ATA TGC ACC AAC TGT TGC GCA GCC Ala Glu Glu Lys Lys Asn Asp Arg Ile
Cys Thr Asn Cys Cys Ala Gly AAA AAG GGT TGT AAG TAC TTT AGT GAT GAT
GGA ACT TTT GTT TGT GAA Lys Lys Gly Cys Lys Tyr Phe Ser Asp Asp Gly
Thr Phe Val Cys Glu GGA GAG TCT GAT CCT AAA AAT CCA AAG CCC TGT GCT
CGG AAT TGT CAT Gly Glu Ser Asp Pro Lys Asn Pro Lys Ala Cys Pro Arg
Asn Cys Asp GGA AGA ATT GCC TAT GGG ATT TGC CCA CTT TCA GAA GAA AAG
AAG AAT Gly Arg Ile Ala Tyr Gly Ile Cys Pro Leu Ser Glu Glu Lys Lys
Asn GAT CGG ATA TGC ACC AAC TGC TGC GCA GGC AAA AAG GGT TGT AAG TAC
Asp Arg Ile Cys Thr Asn Cys Cys Ala Gly Lys Lys Gly Cys Lys Tyr TTT
AGT GAT GAT GGA ACT TTT GTT TGT GAA GGA GAG TCT GAT CCT AAA Phe Ser
Asp Asp Gly Thr Phe Val Cys Glu Gly Glu Ser Asp Pro Lys AAT CCA AAG
GCT TGT CCT CGG AAT TGT GAT GGA AGA ATT GCC TAT GGG Asn Pro Lys Ala
Cys Pro Arg Asn Cys Asp Gly Arg Ile Ala Tyr Gly ATT TGC CCA CTT TCA
GAA GAA AAG AAG AAT GAT CGG ATA TGC ACA AAC Ile Cys Pro Leu Ser Glu
Glu Lys Lys Asn Asp Arg Ile Cys Thr Asn TGT TGC GCA GGC AAA AAG GGC
TGT AAG TAC TTT AGT GAT GAT GGA ACT Cys Cys Ala Gly Lys Lys Gly Cys
Lys Tyr Phe Ser Asp Asp Gly Thr TTT GTT TGT GAA GGA GAG TCT GAT CCT
AGA AAT CCA AAG CCC TGT CCT Phe Val Cys Glu Gly Glu Ser Asp Pro Arg
Asn Pro Lys Ala Cys Pro CGG AAT TGT GAT GGA AGA ATT GCC TAT GGA ATT
TGC CCA CTT TCA GAA Arg Asn Cys Asp Gly Arg Ile Ala Tyr Gly Ile Cys
Pro Leu Ser Glu GAA AAG AAG AAT GAT CGG ATA TGC ACC AAT TGT TGC GCA
GGC AAG AAG Glu Lys Lys Asn Asp Arg Ile Cys Thr Asn Cys Cys Ala Gly
Lys Lys GGC TGT AAG TAC TTT AGT GAT GAT GGA ACT TTT ATT TGT GAA GGA
GAA Gly Cys Lys Tyr Phe Ser Asp Asp Gly Thr Phe Ile Cys Glu Gly Clu
TCT GAA TAT GCC AGC AAA GTG GAT GAA TAT GTT GGT GAA GTG GAG AAT Ser
Glu Tyr Ala Ser Lys Val Asp Glu Tyr Val Gly Glu Val Glu Asn GAT CTC
CAG AAG TCT AAG GTT GCT GTT TCC Asp Leu Gln Lys Ser Lys Val Ala Val
Ser
[0014] This is done, however, with the understanding that the
present invention extends to an equivalent or substantially similar
nucleic acid molecule from any other plant. By "equivalent" and
"substantially similar" is meant at the level of nucleotide
sequence, amino acid sequence, antibody reactivity, monomer
composition and/or processing of the precursor to produce monomers.
For example, a nucleotide sequence having a percentage sequence
similarity of at least 55%, such as about 60-65%, 70-75%, 80-85%
and over 90% when compared to the sequence of SEQ ID NO. 1 would be
considered "substantially similar" to the subject nucleic acid
molecule provided that such a substantially similar sequence
encodes a PI precursor having at least three monomers and
preferably four, five or six monomers as hereinbefore
described.
[0015] In a particularly preferred embodiment, the nucleic acid
molecule further encodes a signal sequence 5' to the open reading
frame and/or a nucleotide sequence 3' of the coding region
providing a full nucleotide sequence as follows (SEQ ID NO. 2):
TABLE-US-00002 CGAGTAAGTA TGGCTGTTCA CAGAGTTAGT TTCCTTGCTC
TCCTCCTCTT ATTTGGAATG TCTCTGCTTG TAAGCAATGT GGAACATGCA GATGCC AAG
GCT TGT ACC TTA AAC Lys Ala Cys Thr Leu Asn TGT GAT CCA AGA ATT GCC
TAT GGA GTT TGC CCG CGT TCA GAA GAA AAG Cys Asp Pro Arg Ile Ala Tyr
Gly Val Cys Pro Arg Ser Glu Glu Lys AAG AAT GAT CGG ATA TGC ACC AAC
TGT TGC GCA GGC ACG AAG GGT TGT Lys Asn Asp Arg Ile Cys Thr Asn Cys
Cys Ala Gly Thr Lys Gly Cys AAG TAC TTC AGT GAT GAT GGA ACT TTT GTT
TGT GAA GGA GAG TCT GAT Lys Tyr Phe Ser Asp Asp Gly Thr Phe Val Cys
Glu Gly Glu Ser Asp CCT AGA AAT CCA AAG GCT TGT ACC TTA AAC TGT GAT
CCA AGA ATT GCC Pro Arg Asn Pro Lys Ala Cys Thr Leu Asn Cys Asp Pro
Arg Ile Ala TAT GGA GTT TGC CCG CGT TCA GAA GAA AAG AAG AAT GAT CGG
ATA TGC Tyr Gly Val Cys Pro Arg Ser Glu Glu Lys Lys Asn Asp Arg Ile
Cys ACC AAC TGT TGC GCA GGC ACG AAG GGT TGT AAG TAC TTC AGT GAT GAT
Thr Asn Cys Cys Ala Gly Thr Lys Gly Cys Lys Tyr Phe Ser Asp Asp GGA
ACT TTT GTT TGT GAA GGA GAG TCT GAT CCT AGA AAT CCA AAG GCT Gly Thr
Phe Val Cys Glu Gly Glu Ser Asp Pro Arg Asn Pro Lys Ala TGT CCT CGG
AAT TGC GAT CCA AGA ATT GCC TAT GGG ATT TGC CCA CTT Cys Pro Arg Asn
Cys Asp Pro Arg Ile Ala Tyr Gly Ile Cys Pro Leu GCA GAA GAA AAG AAG
AAT GAT CGG ATA TGC ACC AAC TGT TGC GCA GGC Ala Glu Glu Lys Lys Asn
Asp Arg Ile Cys Thr Asn Cys Cys Ala Gly AAA AAG GGT TGT AAG TAC TTT
AGT GAT GAT GGA ACT TTT GTT TGT GAA Lys Lys Gly Cys Lys Tyr Phe Ser
Asp Asp Gly Thr Phe Val Cys Glu GGA GAG TCT GAT CCT AAA AAT CCA AAG
GCC TGT CCT CGG AAT TGT GAT Gly Clu Ser Asp Pro Lys Asn Pro Lys Ala
Cys Pro Arg Asn Cys Asp GGA AGA ATT GCC TAT GGG ATT TGC CCA CTT TCA
GAA GAA AAG AAG AAT Gly Arg Ile Ala Tyr Gly Ile Cys Pro Leu Ser Glu
Glu Lys Lys Asn GAT CGG ATA TGC ACC AAC TGC TGC GCA GGG AAA AAG GGT
TGT AAG TAC Asp Arg Ile Cys Thr Asn Cys Cys Ala Gly Lys Lys Gly Cys
Lys Tyr TTT AGT GAT GAT GGA ACT TTT GTT TGT GAA GGA GAG TCT GAT CCT
AAA Phe Ser Asp Asp Gly Thr Phe Val Cys Clu Gly Glu Ser Asp Pro Lys
AAT CCA AAG GCT TGT CCT CGG AAT TGT GAT GGA AGA ATT GCC TAT GGG Asn
Pro Lys Ala Cys Pro Arg Asn Cys Asp Gly Arg Ile Ala Tyr Gly ATT TGC
CCA CTT TCA GAA GAA AAG AAG AAT GAT CGG ATA TGC ACA AAC Ile Cys Pro
Leu Ser Glu Glu Lys Lys Asn Asp Arg Ile Cys Thr Asn TGT TGC GCA GGC
AAA AAG GGC TGT AAG TAC TTT AGT GAT GAT GGA ACT Cys Cys Ala Gly Lys
Lys Gly Cys Lys Tyr Phe Ser Asp Asp Gly Thr TTT GTT TGT GAA GGA GAG
TCT GAT CCT AGA AAT CCA AAG GCC TGT CCT Phe Val Cys Glu Gly Glu Ser
Asp Pro Arg Asn Pro Lys Ala Cys Pro CGG AAT TGT GAT GGA AGA ATT GCC
TAT GGA ATT TGC CCA CTT TCA GAA Arg Asn Cys Asp Gly Arg Ile Ala Tyr
Gly Ile Cys Pro Leu Ser Glu GAA AAG AAG AAT GAT CGG ATA TGC ACC AAT
TGT TGC GCA GGC AAG AAG Glu Lys Lys Asn Asp Arg Ile Cys Thr Asn Cys
Cys Ala Gly Lys Lys GGC TGT AAG TAC TTT AGT GAT GAT CCA ACT TTT ATT
TGT GAA GGA GAA Gly Cys Lys Tyr Phe Ser Asp Asp Gly Thr Phe Ile Cys
Glu Gly Glu TCT GAA TAT GCC AGC AAA GTG GAT GAA TAT GTT GGT GAA GTG
GAG AAT Ser Glu Tyr Ala Ser Lys Val Asp Glu Tyr Val Gly Glu Val Glu
Asn GAT CTC CAG AAG TCT AAG GTT GCT GTT TCC TAAGTCCTAA CTAATAATAT
Asp Leu Gln Lys Ser Lys Val Ala Val Ser GTAGTCTATG TATGAAACAA
AGGCATGCCA ATATGCTCTG TCTTGCCTGT AATCTGTAAT ATGGTAGTGG AGCTTTTCCA
CTGCCTGTTT AATAAGAAAT GGACCACTAG TTTCTTTTAG TTAAAAAAAA
AAAAAAAAAA
including substantially similar variants thereof.
[0016] Accordingly, a preferred embodiment of the present invention
provides a nucleic acid molecule comprising a sequence of
nucleotides as set forth in SEQ ID NO. 1 or 2 which encodes or is
complementary to a sequence which encodes a type II serine PI
precursor from Nicotiana alata or having at least 55% similarity to
said precursor or at least one domain therein wherein said
precursor comprises a signal peptide and at least five monomers and
wherein one of said monomers has a chymotrypsin specific site and
four of said monomers have trypsin specific sites.
[0017] In still a more preferred embodiment, the nucleic acid
molecule is a cDNA molecule and comprises a nucleotide sequence
generally as set forth in SEQ ID NO. 1 or 2 or being substantially
similar thereto as hereinbefore defined to the whole of said
sequence or to a domain thereof.
[0018] Another aspect of the present invention is directed to a
nucleic acid molecule comprising a sequence of nucleotides which
encodes or is complementary to a sequence which encodes a single
type. II serine PI having either a chymotrypsin specific site or a
trypsin specific site and wherein said PI is a monomer of a
precursor PI having at least three monomers of which at least one
of said monomers has a chymotrypsin site and the other of said
monomers has a trypsin site. Preferably, however, the precursor has
four, five or six monomers and is as hereinbefore defined. In its
most preferred embodiment, the plant is N. alata (Link et Otto)
having self-incompatibility genotype S.sub.1S.sub.3, S.sub.3S.sub.3
or S.sub.6S.sub.6, and the nucleic acid molecule is isolatable from
or complementary to genetic sequences isolatable from stigmas and
styles of mature plants. The corresponding mRNA is approximately
1.4 kb and the cDNA has six conserved domains wherein the first two
domains are 100% identical and contain chymotrypsin-specific sites
(Leu-Asn). The third, fourth and fifth domains share 95-98%
identity and have sites specific for trypsin (Arg-Asn). A sixth
domain which also has a trypsin specific site has less identity to
the third, fourth and fifth domains (79-90%) due mainly to a
divergent 3' sequence (see Table 1). The preferred PI inhibitor of
the present invention has a molecular weight of approximately 42-45
kDa with an approximately 29 amino acid signal sequence.
[0019] The N-terminal sequence of the monomeric PI is represented
in each of the six repeated domains in the predicted sequence of
the PI precursor protein. Thus, it is likely that the PI precursor
protein is cleaved at six sites to produce seven peptides. Six of
the seven peptides, peptides 2, 3, 4, 5, 6 and 7 (FIG. 1, residues
25-82 [SEQ ID NO. 5], 83-140 [SEQ ID NO. 6], 141-198 [SEQ ID NO.
7], 199-256 [SEQ ID NO. 8], 257-314 [SEQ ID NO. 9] and 315-368 [SEQ
ID NO. 9], respectively), would be in the same molecular weight
range as the monomeric PI (about 6 kDa) and would have the same
N-terminal sequence. Peptide 7 does not contain a consensus site
for trypsin or chymotrypsin. Peptide 1 (residues 1-24 [SEQ ID NO.
4], FIG. 1) is smaller than 6 kD, has a different N-terminus and
was not detected in a purified monomeric PI preparation. It could
be envisaged that peptide 1 and peptide 7 would form a functional
proteinase inhibitor with the inhibitory site on peptide 1 held in
the correct conformation by disulphide bonds formed between the two
peptides.
[0020] Although not intending to limit the present invention to any
one hypothesis, the PI precursor may be processed by a protease
responsible, for example, for cleavage of an Asn-Asp linkage, to
produce the bioactive monomers. More particularly, the protease
sensitive sequence is R.sub.1-X.sub.1-X.sub.2-Asn-Asp-R.sub.2 where
R.sub.1, R.sub.2, X.sub.1 and X.sub.2 are defined below. The
discovery of such a sequence will enable the engineering of
peptides and polypeptides capable of being processed in a plant by
cleavage of the protease sensitive sequence. According co this
aspect of the present invention there is provided a protease
sensitive peptide comprising the amino acid sequence:
-X.sub.1-X.sub.2-Asn-Asp- wherein X.sub.1 and X.sub.2 are any amino
acid but are preferably both Lys residues. The protease sensitive
peptide may also be represented as:
R.sub.1-X.sub.1-X.sub.2-Asn-Asp-R.sub.2 wherein X.sub.1 and X.sub.2
are preferably the same and are preferably both Lys residues and
wherein R.sub.1 and R.sub.2 are the same or different, any D or L
amino acid, a peptide, a polypeptide, a protein, or a non-amino
acid moiety or molecule such as, but not limited to, an alkyl (eg
methyl, ethyl), substituted alkyl, alkenyl, substituted alkenyl,
acyl, dienyl, arylalkyl, arylalkenyl, aryl, substituted aryl,
heterocyclic, substituted heterocyclic, cycloalkyl, substituted
cycloalkyl, halo (e.g. Cl, Br, I, F), haloalkyl, nitro, hydroxy,
thiol, sulfonyl, carboxy, alkoxy, aryloxy and alkylaryloxy group
and the like as would be apparent to one skilled in the art. By
alkyl, substituted alkyl, alkenyl and substituted alkenyl and the
like is meant to encompass straight and branched molecules, lower
(C.sub.1-C.sub.6) and higher (more than C.sub.6) derivatives. The
term "substituted" includes all the substituents set forth
above.
[0021] In its most preferred embodiment, the protease sensitive
peptide is: R.sub.1--X.sub.1--X.sub.2-Asn-Asp-R.sub.2 wherein
R.sub.1 and R.sub.2 are the same or different and are peptides or
polypeptides and wherein X.sub.1 and X.sub.2 are both Lys
residues.
[0022] Such a protease sensitive peptide can be placed between the
same or different monomers so that upon expression in a suitable
host or in vita the larger molecule can be processed to the
peptides located between the protease sensitive peptides.
[0023] The present invention also extends to a nucleic acid
molecule comprising a sequence of nucleotides which encodes or is
complementary to a sequence which encodes a protease sensitive
peptide comprising the sequence: -X.sub.1-X.sub.2-Asn-Asp- wherein
X.sub.1 and X.sub.2 are preferably the same and are most preferably
both Lys residues. Such a nucleic acid molecule may be part of a
larger nucleotide sequence encoding, for example, a precursor
polypeptide capable of being processed via the protease sensitive
sequence into individual peptides or monomers.
[0024] The protease sensitive peptide of the present invention is
particularly useful in generating poly and/or multi-valent
"precursors" wherein each monomer is the same or different and
directed to the same or different activities such as anti-viral,
anti-bacterial, anti-fungal, anti-pathogen and/or anti-predator
activity.
[0025] Although not wishing to limit this aspect of the invention
to any one hypothesis or proposed mechanism of action, it is
believed that the protease acts adjacent the Asn residue as more
particularly between the Asn-Asp residues.
[0026] The present invention extends to an isolated type II serine
PI precursor from a plant wherein said precursor comprises at least
three PI monomers and wherein at least one of said monomers has a
chymotrypsin specific site and at least one other of said monomers
has a trypsin specific site. Preferably, the PI precursor has four,
five or six monomers and is encoded by the nucleic acid molecule as
hereinbefore described. The present invention also extends to the
individual monomers comprising the precursor. The present invention
also extends to a hybrid recombinant PI precursor molecule
comprising at least two monomers from different PIs as hereinbefore
described.
[0027] The isolated PI or PI precursor may be in recombinant form
and/or biologically pure. By "biologically pure" is meant a
preparation of PI, PI precursor and/or any mixtures thereof having
undergone at least one purification step including ammonium
sulphate precipitation, Sephadex chromatography and/or affinity
chromatography. Preferably, the preparation comprises at least 20%
of the PI, PI precursor or mixture thereof as determined by weight,
activity antibody, reactivity and/or amino acid content. Even more
preferably, the preparation comprises 30-40%, 50-60% or at least
80-90% of PI, PI precursor or mixture thereof.
[0028] The PI or its precursor may be naturally occurring or be a
variant as encoded by the nucleic acid variants referred to above.
It may also contain single or multiple substitutions, deletions
and/or additions to its amino acid sequence or to non-proteinaceous
components such as carbohydrate and/or lipid moieties.
[0029] The recombinant and isolated PI, PI precursor and mixtures
thereof are useful as laboratory reagents, in the generation of
antibodies, in topically applied insecticides as well as orally
ingested insecticides.
[0030] The recombinant PI or PI precursor may be provided as an
insecticide alone or in combination with one or more carriers or
other insecticides such as the BT crystal protein.
[0031] The PI of the present invention is considered to have a
defensive role in organs of the plant, for example, the stigma,
against the growth or infection by pests and pathogens such as
fungi, bacteria and insects. There is a need, therefore, to develop
genetic constructs which can be used to generate transgenic plants
capable of expressing the PI precursor where this can be processed
into monomers of a monomeric PI itself.
[0032] Accordingly, another aspect of the present invention
contemplates a genetic construct comprising a nucleic acid molecule
comprising a sequence of nucleotides which encodes or is
complementary to a sequence which encodes a type II serine PI
precursor or monomer thereof from a plant wherein said precursor
comprises at least three PI monomers and wherein at least one of
said monomers has a chymotrypsin specific site and at least one of
said other monomers has a trypsin specific site and said genetic
sequence further comprises expression means to permit expression of
said nucleic acid molecule, replication means to permit replication
in a plant cell or, alternatively, integration means, to permit
stable integration of said nucleic acid molecule into a plant cell
genome. Preferably, the expression is regulated such as
developmentally or in response to infection such as being regulated
by an existing PI regulatory sequence. Preferably, the expression
of the nucleic acid molecule is enhanced to thereby provide greater
endogenous levels of PI relative to the levels in the naturally
occurring plant. Alternatively, the PI precursor cDNA of the
present invention is usable to obtain a promoter sequence which can
then be used in the genetic construct or to cause its manipulation
to thereby permit over-expression of the equivalent endogenous
promoter. In another embodiment the PI precursor is a hybrid
molecule as hereinbefore described.
[0033] Yet another aspect of the present invention is directed to a
transgenic plant carrying the genetic sequence and/or nucleic acid
molecule as hereinbefore described and capable of producing
elevated, enhanced or more rapidly produced levels of PI and/or PI
precursor or hybrid PI precursor when required. Preferably, the
plant is a crop plant or a tobacco plant but other plants are
usable where the PI or PI precursor nucleic acid molecule is
expressable in said plant. Where the transgenic plant produces PI
precursor, the plant may or may not further process the precursor
into monomers. Alternatively, the genetic sequence may be part of a
viral or bacterial vector for transmission to an insect to thereby
control pathogens in insects which would consequently interrupt the
transmission of the pathogens to plants.
[0034] In still yet another aspect of the present invention, there
is provided antibodies to the PI precursor or one or more of its
monomers. Antibodies may be monoclonal or polyclonal and are useful
in screening for PI or PI precursor clones in an expression library
or for purifying PI or PI precursor in a fermentation fluid,
supernatant fluid or plant extract.
[0035] The genetic constructs of the present invention can also be
used to populate the gut of insects to act against the insect
itself or any plant pathogens therein or to incorporate into the
gut of animals to facilitate the digestion of plant material.
[0036] The present invention is further described by reference to
the following non-limiting Figures and Examples.
[0037] In the Figures:
[0038] FIG. 1 shows the nucleic acid sequence (SEQ ID NO. 2) of the
pNA-PI-2 insert and the corresponding amino acid sequence (SEQ ID
NO. 3) of the N. alata PI protein. The amino acid sequence is
numbered beginning with 1 for the first amino acid of the mature
protein. The signal sequence is encoded by nucleotides 1 to 97 and
the amino acid residues have been assigned negative numbers. The
reactive site residues of the inhibitor are boxed. The N. alata PI
sequence contains six similar domains (domain 1, residues 1 to 58,
domain 2, residues 59-116, domain 3, residues 117-174, domain 4,
residues 175-232, domain 5, residues 233-290 and domain 6, residues
291-343).
[0039] FIG. 2 is a photographic representation showing a gel blot
analysis of RNA from various organs of N. alata Gel Blot of RNA
isolated from organs of N. alata and from stigmas and styles of N.
tabacum and N. sylvestris, hybridised with the cDNA clone NA-PI-2.
St, stigma and style; Ov, ovaries; Po, pollen; Pe, petals; Se,
sepals; L, non-wounded leaves; L4, leaves 4 h after wounding; L24,
leaves 24 h after wounding; Nt, N. tabacum stigma and style; Ns, N.
sylvestris stigma and style; Na HindIII restriction fragments of
Lambda-DNA
[0040] The NA-PI-2 clone hybridised to 2 mRNA species (1.0 and 1.4
kb). The larger mRNA was predominant in stigma and styles, whereas
the smaller mRNA species was more dominant in other tissues. After
high stringency washes, the 1.0 kb mRNA from stigma and style no
longer hybridises to the NA-PI-2 probe.
[0041] FIG. 3 is a photographic presentation depicting in situ
localisation of RNA homologous to NA-PI-2 in stigma and style.
[0042] (a) Autoradiograph of a longitudinal cryosection through the
stigma and style of a 1 cm long bud after hybridisation with the
.sup.32P-labelled NA-PI-2 cDNA probe. [0043] (b) The same section
as (a), stained with toluidine blue. c, cortex; v, vascular
bundles; tt, transmitting tract; s, stigmatic tissue.
[0044] The cDNA probe labelled the cells of the stigma heavily and
some hybridisation to the vascular bundles can be seen. There was
no hybridisation to the epidermis, cortical tissue or transmitting
tissue. Scale bars=200 .mu.m.
[0045] FIG. 4 is a photographic representation of a gel blot
analysis of genomic DNA of N. alata Gel blot analysis of N. alata
genomic DNA digested with the restriction enzymes EcoRI or HindIII,
and probed with radiolabelled NA-PI-2. Size markers (kb) are HinIII
restriction fragments of Lambda-DNA.
[0046] EcoI produced two hybridising fragments (11 kb and 7.8 kb),
while HindIII gave three large hybridising fragments (16.6, 13.5
and 10.5 kb). The NA-PI-2 clone appears to belong to a small
multigene family consisting of at least two members.
[0047] FIG. 5 is a graphic representation of PI activity in various
organs of N. alata Buffer soluble extracts from various organs were
tested for their ability to inhibit trypsin and chymotrypsin.
Stigma and sepal extracts were the most effective inhibitors of
both trypsin (A) and chymotrypsin (B).
[0048] FIG. 6 depicts the steps of the purification of PI from N.
alata stigmas. [0049] (a) Sephadex G-50 gel filtration
chromatography of ammonium sulphate precipitated proteins from
stigma extracts. The PI activity eluted late in the profile. [0050]
(b) 20% w/v SDS-polyacrylamide gel (Laemmli, 1970) of fractions
across the gel filtration column. The gel was silver stained and
molecular weight markers (Pharmacia peptide markers) are in
kilodaltons. A protein of about 6 kD (arrowed) coelutes with the
proteinase inhibitor activity. [0051] (c) Analysis of PI-containing
fractions at different stages of the purification procedure, by
SDS-PAGE. Lane 1, crude stigma extract (5.mu.g); Lane 2, stigma
proteins precipitated by 80% w/v ammonium sulphate (5.mu.g); Lane
3, PI protein eluted from the chymotrypsin affinity column (1
.mu.g).
[0052] The PI is a 6 kD protein and is a major component in
unfractionated buffer soluble extracts from stigmas.
[0053] FIG. 7 is a graphical representation showing hydropathy
plots of the PI proteins encoded by the NA-PI-2 clone from N. alata
and the potato and tomato PI II cDNAs. Values above the line denote
hydrophobic regions and values below the line denote hydrophilic
regions. The putative signal peptides are shaded. The
hydrophobicity profile was generated using the predictive rules of
Kyte and Doolittle (1982) and a span of 9 consecutive amino acids.
[0054] (a) Hydropathy profile of the N. alata PI protein. The six
repeated domains in the predicted precursor protein are labelled
I-VI. The hydrophilic regions containing the putative cleavage
sites for production of the 6 kD PI species are arrowed. The
regions corresponding to the peptides that would be produced by
cleavage at these sites are marked C for chymotrypsin inhibitor, T
for trypsin inhibitor and x for the two flanking peptides. [0055]
(b) Hydropathy profile of the potato PI II protein.
(Sanchez-Serrano et al., 1986). The two repeated domains in the PI
II protein are labelled I and II. The putative cleavage sites for
production of PCI-1 are arrowed (Hass et al., 1982) and the region
spanned by PCI-1 is marked. [0056] (c) Hydropathy profile of the
polypeptide encoded by the tomato PI II cDNA. (Graham et al.,
1985). The two domains are labelled, I and II and the residues
which would be potential processing sites are arrowed. These sites
are not present in regions predicted to be hydrophilic and
consequently a cleavage product is not marked.
[0057] FIG. 8 shows an immunoblot analysis of the PI protein in
stigmas of developing flowers. [0058] (a) Developing flowers of N.
alata [0059] (b) SDS-PAGE of stigma proteins at the stages of
development shown in (a) 5 .mu.g of each extract was loaded. The
peptide gel was silver stained and molecular weight markers (LKB
Low Molecular weight and Pharmacia peptide markers) are in
kilodaltons. [0060] (c) Immunoblot of a gel identical to (b),
probed with anti-PI antiserum.
[0061] Stigmas from developing flowers contain four proteins of
approximately 42 kD, 32 kD, 18 kD and 6 kD that bind to the anti-PI
antibody. The 42 kD and the 18 kD components decrease in
concentration as the flowers mature, while the 6 kD PI protein
reaches a maximum concentration just before anthesis. The level of
the 32 kD component, which runs as a doublet, does not alter
significantly during flower development.
[0062] FIG. 9 shows the separation and identification of the 6 kD
proteinase inhibitor species from N. alata stigmas
A. Separation of the 6 kD PIs by Reversed Phase HPLC
Chromatography
[0063] Four major peaks were obtained with retention times of about
15.5 min(peak1), 20.5 min(peak2), 22.5 min(peak3), 24 min(peak4).
The peptides in each peak have been identified by a combination of
N-terminal analysis and mass spectrometry. See B for description of
C1 and T1-T4.
[0064] B. The five homologous peptides produced from the PI
precursor protein: C1, chymotrypsin inhibitor, T1-T4 trypsin
inhibitors. The solid bars represent the reactive sites of the
inhibitors. The precursor protein is drawn minus the signal
sequence. region of the six repeated domains (amino acids 1-343,
FIG. 1). non-repeated sequence (amino acids 344-368, FIG. 1). The
arrows point to the processing sites in the precursor protein.
[0065] C. The amino acid sequence of C1 and T1-T4 predicted from
the cDNA clone and confirmed by N-terminal sequencing of the
purified peptides. The amino acid at the carboxy-terminus of each
peptide was obtained by accurate mass determination using an
electro-spray mass spectrometer. The C1 and T1 inhibitors differ by
five amino acids (bold). Two of these amino acids are located at
the reactive site (underlined) and the other two to three reside at
the carboxy-terminus. Peptides T2-T4 have changes in three amino
acids (boxed) that are conserved between C1 and T1. Peptides T2 and
T3 are identical to each other. Mass spectrometry was used to
demonstrate that other forms of C1 and T1-T4 occur due to
non-precise trimming at the N- and C-termini. That is, some forms
are missing residue 1 or residue 53 and others are missing both
residue 1 and 53 (see Table 2).
[0066] FIG. 10 shows the amino-acid sequence around the processing
sites in the precursor PI protein.
[0067] The sequence in bold is the amino-terminal sequence obtained
from the purified PI protein. The sequence labelled with negative
numbers is the flanking sequence predicted from the cDNA clone. The
predicted precursor protein contains six repeats of this
sequence.
[0068] FIG. 11 shows the PI precursor produced in a baculovirus
expression system and the products obtained after digestion of the
affinity purified PI precursor by the endoproteinase Asp-N.
A. The PI Precursor Produced by the Recombinant Baculovirus.
[0069] Immunoblot containing affinity and HPLC purified PI
precursor from N. alata stigmas at the green bud stage of
development (lane 1) and affinity purified PI precursor produced by
the recombinant baculovirus (lane 2). Proteins were fractionated by
electrophoresis on a 15% w/v SDS-polyacrylamide gel prior to
electrophoretic transfer to nitrocellulose. The blot was incubated
with the antibody raised in rabbits to the 6 kD PI species from
stigmas. The recombinant virus produced an immunorective protein of
42 kD that is the same size as the PI precursor protein produced by
stigmas (arrowed).
B. Cleavage of the PI Precursor by Endoproteinase Asp-N.
[0070] 15% SDS-polyacrylamide gel stained with silver containing:
1, PI precursor, produced by baculovirus, incubated without enzyme,
2, enzyme incubated without precursor. 6 kD, PI peptides of about 6
kD purified from N. alata stigmas. 1 m, 5 m, 30 m, reaction
products produced after 1, 5 and 30 minutes of incubation. 2 h and
24 h, reaction products after 2 and 24 h of incubation. Peptides of
about 6-7 kD were detected within one minute of incubation of the
precursor with the enzyme. After 24 h only peptides of 6-7 kD were
detected. The bands smaller than 42 kD in track 1 are due to
truncated forms of the precursor produced by premature termination
of translation in the baculovirus expression system.
[0071] FIG. 12 Preparative chromatography by reversed phase HPLC of
the peptides produced from the precursor by Asp-N digestion
[0072] HPLC profile of peptides produced by Asp-N digestion of the
PI precursor. The major peaks had a retention time of 19 min
(termed Asp-N1) and 21 min (termed Asp-N2). The peptides in these
peak fractions (1 & 2) had a slightly slower mobility on
SDS-PAGE than the 6 kD peptides from stigmas (C, inset). The
proteinase inhibitory activity of Asp-N1 and Asp-N2 was tested
against trypsin and chymotrypsin.
[0073] FIG. 13 shows a comparison of the trypsin and chymotrypsin
inhibition activity of the PI precursor, PI peptides from stigmas
and in vitro produced PI peptides from the PI precursor.
[0074] PI precursor or PI peptides (0-1.0.mu.g) were tested for
their ability to inhibit 1.0 .mu.g of trypsin or chymotrypsin as
described in the materials and methods. Inhibitory activity is
expressed as the percentage of proteinase activity remaining after
the proteinase had been preincubated with the PI with 100%
remaining activity taken as the activity of the proteinase
preincubated with no PI. Experiments were performed in duplicate
and mean values were plotted. Deviation from the mean was 8% or
less.
[0075] FIG. 14 is a graphical representation showing a growth curve
for T. commodus nymphs reared on control artificial diet, soybean
Bowman-Birk inhibitor and N. alata PI. The vertical axis represents
the mean weight of the crickets in each treatment (+/-standard
error) in mg. The horizontal axis represents the week number. The
crickets reared on the N. alata PI showed a lower mean weight than
those reared on both the control diet and the diet containing the
soybean inhibitor, throughout the experiment. TABLE-US-00003
SUMMARY OF SEQ ID NOs SEQ ID NO. 1 Nucleotide coding region of N.
alata PI precursor SEQ ID NO. 2 Full length nucleotide sequence of
N. alata PI precursor SEQ ID NO. 3 Amino acid sequence
corresponding to SEQ ID NO. 1 SEQ ID NO. 4 Residues 1-24 of SEQ ID
NO. 2 (peptide 1) SEQ ID NO. 5 Residues 25-82 of SEQ ID NO. 2
(peptide 2) SEQ ID NO. 6 Residues 83-140 of SEQ ID NO. 2 (peptide
3) SEQ ID NO. 7 Residues 141-198 of SEQ ID NO. 2 (peptide 4) SEQ ID
NO. 8 Residues 199-256 of SEQ ID NO. 2 (peptide 5) SEQ ID NO. 9
Residues 257-314 of SEQ ID NO. 2 (peptide 6) SEQ ID NO. 10 Residues
315-368 of SEQ ID NO. 2 (peptide 7) SEQ ID NO. 11 N-terminal amino
acid sequence of 6 kD PI protein SEQ ID NO. 12 N-terminal amino
acid sequence of 6 kD PI protein
EXAMPLE 1
1. Materials and Methods
Plant Material
[0076] Nicotiana alata (Link et Otto) plants of
self-incompatibility genotype S.sub.1S.sub.3, S.sub.3S.sub.3 and
S.sub.6S.sub.6 were maintained under standard glasshouse conditions
as previously described (Anderson et al., 1989). Organs were
collected directly into liquid Nitrogen to avoid induction of a
wound response and stored at -70.degree. until required. To study
the effect of wounding on gene expression, leaves were wounded by
crushing across the mid-vein with a dialysis clip. Leaves were
collected 4 and 24 hours after wounding.
Identification and Sequencing of a cDNA Clone Encoding PI
[0077] Polyadenylated RNA was prepared from stigmas and styles,
isolated from mature flowers of N. alata (genotype S.sub.3S.sub.3),
and used to construct a cDNA library in Lambda gt10 (Anderson et
al., 1989). Single stranded .sup.32P-labelled cDNA was prepared
from mRNA from stigmas and styles of N. alata (genotype
S.sub.3S.sub.3 and S.sub.6S.sub.6) and used to screen the library
for highly expressed clones which were not S-genotype specific
(Anderson et al., 1989). Plaques which hybridised strongly to cDNA
probes from both S-genotypes were selected and assembled into
groups on the basis of cross-hybridisation. The longest clone of
each group was subcloned into M13mp18 and pGEM 3zf+, and sequenced
using an Applied Biosystems Model 373A automated sequencer. Both
dye primer and dye terminator cycle sequencing chemistries were
performed according to standard Applied Biosystems protocols.
Consensus sequences were generated using SeqEd.TM. sequence editing
software (Applied Biosystems). The GenBank database was searched
for sequences homologous to these clones. Because of the high
degree of sequence similarity between the six domains of the N.
alata PI clone, sequencing primers were made to non-repeated 3'
sequences (nucleotides 1117-1137, 1188-1203 and 1247-1267), and to
a 5' sequence before the start of the repetitive regions
(nucleotides 74-98). In addition, the pNA-PI-2 insert was
restricted with endonuclease HaeIII, which cut at nucleotides 622
and 970 to produce three fragments. The fragments were subcloned
into pGEM7zf+ and sequenced in both directions, using the M13
forward and reverse primers. The repetitive nature of the pNA-PI-2
insert rendered it unstable in both phagemid and plasmid vectors
when cultures were grown longer than 6 hours.
RNA Gel Blot Analysis
[0078] Total RNA was isolated and separated on a 1.2% w/v
agarose/formaldehyde gel as previous described (Anderson et al.,
1989). The RNA was transferred to Hybond-N (Amersham) and probed
with the insert from pNA-PI-2 labelled with .sup.32P using random
hexanucleotides (1.times.10.sup.8 cpm .mu.g.sup.-1;
1.times.10.sup.7 cpm ml.sup.-1)(Feinberg and Vogelstein, 1983).
Prehybridisation and hybridisation, at 68.degree. C., were as
described by Anderson et al. (1989). The filters were washed in
2.times.SSC, 0.1% w/v SDS or 0.2.times.SSC, 1% w/v SDS at
68.degree. C.
In Situ Hybridisation
[0079] In situ hybridisation was performed as described by Cornish
et al., 1987. The probe was prepared by labelling the insert from
pNA-PI-2 (100 ng) to a specific activity of 10.sup.8 cpm
.mu.g.sup.-1 by random hexanucleotide priming (Feinberg and
Vogelstein, 1983). The labelled probe was precipitated, and
resuspended in hybridisation buffer (50 .mu.l), and 5 .mu.l was
applied to the sections. The sections were covered with coverslips,
and incubated overnight at 40.degree. C. in a closed box containing
50% v/v formamide. After incubation, sections were washed
sequentially in 4.times.SSC at room temperature, 2.times.SSC at
room temperature, and 1.times.SSC at 40.degree. C. for 40 min. The
slides were dried and exposed directly to X-ray film (Cronex MRF
32, Dupont) at room temperature, overnight. Hybridised sections
were counterstained with 0.025% w/v toluidine blue in H.sub.2O, and
mounted in Eukitt (Carl Zeiss, Freilburg, FRG). Autoradiographs
were transposed over sections to give the composites shown.
DNA Gel Blot Analysis
[0080] Genomic DNA was isolated from young leaves of N. alata by
the procedure of Bernatzky and Tanksley (1986). DNA (10 .mu.g) was
digested to completion with the restriction endonucleases EcoRI or
HindIII, separated by electrophoresis on a 0.9% w/v agarose gel,
and transferred to Hybond-N (Amersham) by wet blotting in
20.times.SSC. Filters were probed and washed as described for RNA
blot analysis.
Preparation of Protein Extracts
[0081] Soluble proteins were extracted from plant material by
freezing the tissue in liquid N.sub.2, and grinding to a fine
powder in a mortar and pestle. The powdered tissue was extracted in
a buffer consisting of 100 mM Tris-HCl, pH 8.5, 10 mM EDTA, 2 mM
CaCl.sub.2, 14 .mu.M .beta.-mercaptoethanol. Insoluble material was
removed by centrifugation at 10,000 g for 15 min. Protein
concentrations were estimated by the method of Bradford (1976) with
Bovine Serum Albumin (BSA) as a standard.
Proteinase Inhibition Assays
[0082] Protein extracts and purified protein were assayed for
inhibitory activity against trypsin and chymotrypsin as described
by Rickauer et al (1989). Inhibitory activity was measured against
1 .mu.g of trypsin (TPCK-treated; Sigma) or 3 .mu.g of chymotrypsin
(TLCK-treated; Sigma). The rate of hydrolysis of synthetic
substrates N-.alpha.-P-tosyl-L-arginine methyl ester (TAME) and
N-benzoyl-L-tyrosine ethyl ester (BTEE) by trypsin and
chymotrypsin, respectively, were taken as the uninhibited activity
of the enzymes. Inhibitory activity of the extract was expressed as
the percentage of control protease activity remaining after the
protease had been pre-incubated with the extract. The PI peptides
from stigma, PI precursor and Asp-N processed peptides were assayed
for inhibitory activity as described by Christeller et al
(1989).
Purification of the N. alata PI Protein
[0083] Stigmas (1000; 10 g) were ground to a fine powder in liquid
N.sub.2, and extracted in buffer (100 mM Tris-HCl, pH8.5, 10 mM
EDTA, 2 mM CaCl.sub.2, 14 .mu.M .beta.-mercaptoethanol, 4 ml/g
tissue). To concentrate the extract prior to the first purification
step, gel filtration, the inhibitory activity was precipitated with
80% w/v ammonium sulphate, the concentration required to
precipitate all the proteinase inhibitory activity.
[0084] The ammonium sulphate pellet was resuspended in 5 ml of
0.15M KCl, 10 mM Tris-HCl, pH 8.1, and loaded onto a Sephadex G-50
column (2 cm.times.100 cm) equilibrated with the same buffer. The
fractions (10 ml) eluted from this column and containing proteinase
inhibitory activity were pooled and applied to an affinity column
of Chymotrypsin-Sepharose CL4B [100 mg TLCK-treated
.alpha.-chymotrypsin (Sigma) cross-linked to 15 ml Sepharose CL4B
(Pharmacia) by manufacturers instructions]. The column was washed
with 10 volumes of 0.15M KCl/10 mM Tris-HCl, pH 8.1, prior to
elution of bound proteins with 7 m urea, pH 3 (5 ml fractions). The
eluate was neutralised immediately with 200 .mu.l M Tris-HCl pH 8,
and dialyzed extensively against deionised H.sub.2O.
Amino Acid Sequence Analysis
[0085] Purified PI protein was chromatographed on a reverse phase
HPLC microbore column prior to automated Edman degradation on a gas
phase sequencer (Mau et al., 1986). Phenylthiohydantoin (PTH) amino
acids were analysed by HPLC as described by Grego et al.
(1985).
Production of a Polyclonal Antiserum to the N. alata PI
[0086] The purified proteinase inhibitor (FIG. 6c, lane 3) was
conjugated to a carrier protein, keyhole limpet haemocyanin (KLH)
(Sigma), using glutaraldehyde, as follows. 1 mg of PI protein was
dissolved in 1.5 ml H.sub.2O, and mixed with 0.3 mg KLH in 0.5 ml
of 0.4M phosphate buffer, pH7.5. 1 ml of 20 mM glutaraldehyde was
added dropwise over 5 min, with stirring at room temperature. The
mixture was stirred for 30 min at room temperature, 0.25 ml of
glycine was added, and the mixture was stirred for a further 30
min. The conjugated protein was then dialyzed extensively against
normal saline (0.8% w/v NaCl). The equivalent of 100 .mu.g of PI
protein was used for each injection. Freund's complete adjuvant was
used for the first injection, and incomplete adjuvant for two
subsequent booster injections. The IgG fraction of the antiserum
was separated on Protein A Sepharose (Pharmacia) according to
manufacturer's instructions.
Protein Gel Blot Analysis
[0087] Protein extracts were electrophoresed in 15% w/v
SDS-polyacrylamide gels (Laemmli, 1970) and transferred to
nitrocellulose in 25 mM Tris-HCl, 192 mM glycine, 20% v/v methanol,
using a BioRad Trans-Blot.RTM.Semi-dry electrophoretic transfer
cell (12V, 20 min). Loading and protein transfer were checked by
staining the proteins on the membranes with Ponceau S (Harlow and
Lane, 1988). Membranes were blocked in 3% w/v bovine serum albumin
for 1 h, and incubated with the anti-PI antibody (2 .mu.g/ml in 1%
w/v BSA, Tris Buffered Saline) overnight at room temperature. Bound
antibody was detected using biotinylated donkey anti-rabbit IgG
(1/500 dilution, Amersham) and the Amersham Biotin-Streptavidin
system according to procedures recommended by the manufacturer.
Proteolysis of the PI Precursor by Endoproteinase Asp-N
[0088] Affinity-purified PI precursor (1.25 mg) was incubated at
37.degree. C. with endoproteinase Asp-N (2 .mu.g) in 100 mM
NH.sub.4HCO.sub.3, pH 8.5 in a total volume of 1 ml for 48 h.
Reaction products were separated by reversed-phase HPLC using an
analytical Brownlee RP-300 Aquapore column (C8, 7.mu.m,
4.6.times.100 mm). The column was equilibrated in 0.1% v/v TFA and
peptides were eluted with the following program: 0-25% B (60% v/v
acetonitrile in 0.089% v/v TFA) applied over 5 min, followed by a
gradient of 25-42% B over the next 40 min, and ending with a
gradient of 42-100% B over 5 minutes. The flow rate was 1.0 ml/min
and peptides were detected by absorbance at 215 nm. Each peak was
collected manually and freeze dried. Concentration was estimated by
response obtained with each peak on the UV detector at 215 nm.
2. Cloning of PI Precursor Gene
Isolation and Characterisation of the PI cDNA Clone.
[0089] A cDNA library, prepared from mRNA isolated from the stigmas
and styles of mature flowers of N. alata, was screened for clones
of highly expressed genes which were not associated with
self-incompatibility genotype. Clones encoding a protein with some
sequence identity to the type II proteinase inhibitors from potato
and tomato (Thornburg et at, 1987; Graham et at, 1985) were
selected. The largest clone, NA-PI-2, is 1360 base pairs long with
an open reading frame of 1191 nucleotides. The nucleic acid
sequence (SEQ ID NO. 2) and the predicted amino acid sequence (SEQ
ID NO. 3) of the N. alata clone, NA-PI-2 is shown in FIG. 1. There
are no potential N-glycosylation sites. Surprisingly, the N. alata
cDNA clone encodes a protein with six repeated domains that have
high, but not perfect, sequence identity (FIG. 1). Each of these
domains contains a potential reactive site which is highlighted in
FIG. 1. The residues at the putative reactives sites of the N.
alata PI are consistent with the inhibitor having two sites which
would specifically inhibit chymotrypsin (Leu5-Asn6, Leu63-Asn64)
and four sites specific for trypsin (Arg121-Asn122, Arg179-Asn180,
Arg237-Asn238 and Arg295-Asn296).
[0090] To ensure that the repeat structure of NA-PI-2 was not due
to a cloning artifact, three additional cDNA clones were sequenced,
and found to be identical to NA-PI-2.
[0091] Table 1 is a comparison of the percentage amino acid
identity of the six domains of the PI precursor.
Temporal and Spatial Expression of the PI mRNA
[0092] Total RNA, isolated from various tissues of N. alata, was
probed with the PI cDNA clone in the RNA gel blot analyses shown in
FIG. 2. Two hybridising messages of 1.0 and 1.4 kb were present in
total RNA isolated from styles (including stigmas). Only the larger
message, which was predominant in this tissue, is of sufficient
size to encode the cDNA clone NA-PI-2 (1.4 kb). The smaller message
is not detected with the cDNA probe at higher stringency. An
homologous message of approximately 1.4 kb was also present in RNA
isolated from the styles of N. tabacum and N. sylvestris (FIG.
2).
[0093] In the other floral organs (except pollen), both messages
were detectable at low levels, however, the smaller RNA species
appeared more abundant. There was no hybridisation to pollen RNA.
No hybridising species were evident in leaf RNA, but two species,
1.0 and 1.4 kb were detected 24 hours after mechanical wounding.
The smaller message (1.0 kb) was more abundant in this case.
[0094] In situ hybridisation of radiolabelled N. alata PI cDNA to
longitudinal sections of styles from immature (1 cm long) buds is
shown in FIG. 3. RNA homologous to the cDNA clone bound strongly to
cells of the stigma and weakly to vascular bundles. No
hybridisation was detected in the cortical tissue, transmitting
tract tissue, or epidermis of the style. The same pattern of
hybridisation was observed in mature receptive flowers. Control
sections treated with ribonuclease A prior to hybridisation were
not labelled.
Genomic DNA Blot Analysis
[0095] The cDNA clone NA-PI-2, was used as a probe on the DNA gel
blot shown in FIG. 4 which contained genomic DNA, digested with
either EcoI or HindIII. EcoRI produced two hybridising fragments
(11 kb and 7.8 kb) and HindIII produced three large hybridising
fragments (16.6, 13.5 and 10.5 kb).
Distribution of PI Activity in Various Tissues of N. alata
[0096] The inhibition of trypsin and chymotrypsin by crude extracts
of various organs of N. alata is shown in FIG. 5. Stigma extract
was the most effective inhibitor of both trypsin and chymotrypsin.
The stigma extracts had up to eight times more inhibitory activity
than sepal extracts, and more than 20 times more activity than
extracts from styles, petals, leaves and wounded leaves.
Purification of PI from N. alata Stigmas
[0097] Stigmas of N. alata were extracted in buffer and the
inhibitory activity was concentrated by precipitation with 80% w/v
ammonium sulphate. The precipitate was redissolved and fractionated
by gel filtration on Sephadex G-50. Most of the protein in the
extract eluted early in the profile illustrated in FIGS. 6a and 6b,
relative to the proteinase inhibitor. Fractions with proteinase
inhibitor activity were pooled and applied to an affinity column of
chymotrypsin-Sepharose. The PI activity co-eluted with a protein of
about 6 kD, which appeared to migrate as a single band on the 20%
SDS-polyacrylamide gel shown in FIG. 6c. The purity of the PI at
various stages of purification was assessed by SDS-PAGE (FIG. 6c).
The purified inhibitor represented approximately 50% of the
inhibitory activity present in the crude extract.
Amino Acid Sequence of the N-Terminus of the 6 kD PI Protein
[0098] The N-terminal amino acid sequence DRICTNCCAG(T/K)KG (SEQ ID
NO. 11; SEQ ID NO. 12, respectively) was obtained from the purified
PI protein. This sequence of amino acids corresponds to six regions
in the deduced sequence of the cDNA clone, starting at positions
25, 83, 141, 199, 257 and 315 in FIG. 1. At position 11 of the
N-terminal sequence, both threonine and lysine were detected. This
is consistent with the purified inhibitor comprising a mixture of
six peptides beginning with the sequences underlined in FIG. 1, as
the first two peptides contain threonine at this position, while
the other four peptides have lysine at this position. The position
of these peptides relative to the six repeated domains in the
predicted precursor protein is illustrated in FIG. 7. Five of the
six predicted 6 kD peptides, contain a reactive site for either
chymotrypsin or trypsin (FIGS. 1 and 7). The sixth potential
peptide is four amino-acids shorter than the other five peptides
(fifty eight amino-acids) and may not be active, as it does not
contain an inhibitory site. The peptide from the N-terminus (x in
FIG. 7) has a potential chymotrypsin reactive site but is much
shorter (24 amino acids).
Distribution of the PI Protein in N. alata
[0099] A polyclonal antiserum was raised to the purified PI protein
conjugated to keyhole limpet haemocyanin. The antibody reacted
strongly with the purified 6 kD PI protein in immunoblot analyses
and bound only to a 6 kD and a 32 kD protein, which appears as a
doublet, in total stigma and style extracts from mature flowers.
FIG. 8 is an immunoblot containing protein extracts of stigmas from
flowers at different stages of development (1 cm long buds to
mature flowers) probed with the anti-PI antiserum. Larger cross
reacting proteins of approximately 18 kD, and 42 kD were detected
in buds from 1 cm to 5 cm in length in addition to the 6 kD and the
32 kD protein. The 18 kD and 42 kD proteins decreased in
concentration with maturity, while the 6 kD protein reached a peak
concentration just before anthesis. The concentration of the 32 kD
protein remained relatively constant during flower maturation.
TABLE-US-00004 TABLE 1 N. alata PI 1 2 3 4 5 6 N. alata 1 100 88 88
90 79 2 88 88 90 79 3 97 95 86 4 98 90 5 90 6
EXAMPLE 2
Purification and Identification of PI Monomers
1. Materials and Methods
Separation of the 6 kD PI Species by Reversed Phase
Chromatography
[0100] Stigmas (21,000) were ground and extracted as described for
purification of the PI protein. After gel filtration on a Sephadex
G-50 gel filtration column (5 cm.times.800 cm, 3000 stigmas per
separation) the peptides were lyophilized and applied to a Brownlee
RP-300 C8 Reversed-phase column, 10.times.250 mm, on a Beckman HPLC
system Gold, and eluted with 0.1% v/v Trifluoroacetic acid (TFA)
and an acetonitrile gradient (0-10% over 5 mins, 10-25% over 40
mins and 25-60% over 10 mins), at 5 ml/min. Peak fractions,
designated fraction 1, 2, 3 and 4 were collected and freeze
dried.
Electrospray Mass Spectrometry
[0101] On line mass spectrometric analysis of HPLC eluates was
performed by application of 20 pmoles of each PI preparation
(fraction 1, 2, 3 & 4) in 2 .mu.l of water onto a Brownlee
RP-300 C8 reversed-phase column (150.times.0.20 mm internal
diameter fused-silica capillary column) on a modified
Hewlett-Packard model HP1090L liquid chromatograph and elution with
a linear gradient of acetonitrile (0.05% v/v TFA to 0.045% v/v
TFA/60% v/v acetonitrile in 30 min.) at a flow rate of 1 .mu.l/min
and a column temperature of 25.degree. C. The eluant was monitored
at 215 nm using a Spectral Physics forward optics scanning detector
with a 6-mm pathlength U-shaped axial beam capillary flow cell (LC
Packings, Netherlands). Mass spectra were acquired on a
Finnigan-Mat triple quadrupole mass spectrometer (model TSQ-700,
San Jose, Calif.) equipped with an electrospray ionisation (ESI)
source (Analytica, Branford, Conn.). The electrospray needle was
operated in positive ion mode at a voltage differential of -4 kV.
The sheath liquid was 2-methoxyethanol delivered at 1 .mu.l/min via
a syringe drive (Harvard Apparatus, South Natick, Mass.). The
nitrogen drying gas conditions were as follows: heater temperature,
275.degree. C.; pressure, 15 psi; flow rate, -15 stdL/min. The
nitrogen sheath gas was supplied at 33 psi. Gaseous nitrogen was
obtained from a boiling liquid nitrogen source. Peptides were
introduced into the ESI source at 1.0 .mu.l/min by on-line
capillary RP-HPLC as described above. Spectra were acquired
scanning from m/z 400 to 2000 at a rate of 3 sec. Data collection
and reduction were performed on a Dec5100 computer using Finnigan
BIOMASS.TM. software.
2. Results
[0102] Separation and identification of the individual 6 kD PI
species from N. alata stigmas The five of the six peptides of about
6 kD that were predicted to be present in the purified 6 kD PI
preparation have been separated from each other by reversed-phase
HPLC chromatography. Four peaks were obtained (FIG. 9a) and the
peptides within each peak were identified by electrospray mass
spectrometry (Table 2). The peptides have been designated C1, T1,
T2, T3 and T4 according to their position in the PI precursor and
the presence of a chymotrypsin or trypsin reactive site (FIG. 9b).
The first HPLC peak (FIG. 9a) corresponds to the chymotrypsin
inhibitor C1, the second peak is composed of a mixture of T2 and T3
(identical to each other) and T4 that differs from T2 and T3 by one
amino-acid at position 32. The third peak contains the peptide T1
and the fourth peak is composed of a mixture of T1, T2/T3 and T4
(Table 2).
[0103] The site of processing has not been precisely determined,
but is likely to be located between the aspartate (N) and
asparagine (D) residues in the sequence outlined in FIG. 10.
Proteases with specific requirements for asparagine residues have
been isolated from vacuoles from immature soybean seeds and pumpkin
cotyledons (Scott et al., 1992, Hara-Nishimura et al., 1991). This
is consistent with the immunogold localization of the PI in the
vacuoles of the papillae and the underlying secretory cells in the
stigma of N. alata (Atkinson, 1992). In the case of the N. alata
PI, processing analogous to that of peptide hormones is also
possible because each of the possible 6 kD peptides are flanked by
dibasic residues (Lys-Lys, position-2 &-3 in FIG. 10). However,
a system like this has not been described in plants, and it is more
likely that the dibasic residues contribute to the predicted
hydrophilic loops that present the processing site on the surface
of the molecule.
[0104] The data from the mass spectrometric analysis shows that
once the initial cleavage has occurred the new carboxy terminus is
trimmed back (FIG. 10). The EEKKN sequence (SEQ ID NO. 14) is
removed completely but the trimming is not precise, sometimes an
additional amino acid is removed. Steric hindrance probably
prevents further trimming. Occasionally the aspartate is also
removed from the N-terminus.
EXAMPLE 3
[0105] Production of PI Precursor in Insect Cell (Sf9) Culture
Using a Recombinant Baculovirus Vector.
[0106] cDNA encoding the PI precursor (FIG. 1) was inserted into
the Eco R1 site of the plasmid vector pVL 1392, which is the same
as pVL941 (Lucknow and Summers, 1989) except that a multiple
cloning site was inserted at the BamH1 site. The plasmid designated
pRH11, contains the PI cDNA in the correct orientation with respect
to the direction of transcription directed by the polyhedrin
promoter. Recombinant baculovirus was obtained by co-transfection
of Spodoptera frugiperda cells with baculovirus DNA and pRH11. The
recombinant viruses, produced by homologous recombination, were
plaque purified and amplified prior to infection of insect cells
for protein production. All procedures for production of
recombinant baculovirus, titration of the virus and maintenance and
infection of the Sf9 cells were obtained from King and Posse
(1992). For production of the PI precursor, monolayers of Sf9 cells
in large flasks (175 cm.sup.2) were infected at the time of
confluence with an inoculum of high-titre recombinant virus at a
multiplicity of infection of 5-10 pfu/cell. Culture fluid was
collected after 4 days of infection, clarified by centrifugation
and the PI precursor was purified by application to a
Chymotrypsin-Sepharose affinity column as described for the 6 kD PI
species from stigmas. PI precursor eluted from the column in 7M
urea, pH3 was neutralized immediately with 1M Tris-HCl buffer pH8,
dialysed extensively against Milli-Q water, concentrated 20-50 fold
by ultrafiltration using a Diaflow YM10 filter and stored frozen at
-20 C.
[0107] The cDNA clone encoding the PI precursor was engineered into
a baculovuirus vector for the production of the precursor from
infected insect cells. The insect cells produced a 42 kD protein
that cross reacted with the antibodies raised to the 6 kD PI
peptides from stigma and bound to the chymotrypsin affinity column.
This 42 kD protein was identical in size to the 42 kD precursor
produced in the immature stigmas of N. alata (FIG. 11) and had the
N-terminal sequence LysAlaCysThrLeuAsn (SEQ ID NO. 13)
demonstrating that the signal sequence had been processed correctly
by the insect cells (FIG. 1). Based on these results, the 42 kD
protein produced in the baculovirus expression system will now be
referred to as the PI precursor. The 42 kD PI precursor had
inhibitory activity against chymotrypsin but no inhibitory activity
against trypsin (FIG. 13). Processing of the PI precursor by the
endoproteinase AspN led to the production of stable peptides of
about 6 kD that were partially purified by reversed phase HPLC
(FIG. 12). These peptides have equivalent inhibitory activity
against trypsin and chymotrypsin as the 6 kD peptides isolated from
stigma, indicating that processing of the precursor is required to
activate the trypsin inhibitory activity but not all the
chymotrypsin activity. Since AspN cleaves specifically adjacent to
Aspartate residues (between Asn-1 and Asp1 in FIG. 10) and has no
trimming activity, the peptides produced in vitro will be similiar
to those produced in stigmas except for the presence of the
sequence EEKKN (SEQ ID NO. 14) at the C-terminus. This provides
further evidence that precise processing of the N-and C-termini is
not required to obtain an active 6 kD PI peptide. Asp-N1 is more
efficient at inhibiting chymotrypsin than trypsin and is thus
likely to be predominantly a C1 analogue (FIG. 9b). Asp-N2 is a
more efficient trypsin inhibitor and probably contains the T1-T4
analogues.
EXAMPLE 4
[0108] Effect of PIs on protease activity in unfractionated gut
extracts from various insects Activity of PIs on gut proteases was
measured using the procedure of Christeller et al., (1992) as
follows. An aliquot of 1 uM of inhibitor (0-10 .mu.l, at least
5-fold excess over proteases present in the gut) was mixed with 150
.mu.l of 10 mM CAPS buffer, pH 10, and preincubated with each
insect gut extract (0-15 .mu.l), for 20 min at 30.degree. C. The
reaction was started by the addition of 50 .mu.l of
.sup.14C-labelled casein substrate (400 .mu.g protein, specific
activity 25,000-75,000 dpm mg.sup.-1) and continued for 30 min at
30.degree. C. until 50 .mu.l of cold 30% (w/v) TCA was added to
terminate the reaction. After incubation on ice for 30 min,
undigested protein was pelleted by centrifugation at 20.degree. C.
for 5 min at 10,000 g. The supernatant was removed, mixed with
scintillation fluid and the radioactivity measured. Assays were
performed at pH 10 except for L. sericata and C. rufifacies when 10
mM Tris-HCl, pH 8.0 was used.
[0109] Table 3 shows the inhibitory activity of the pooled 6 kD PI
peptides (C1, T1, T2/T3, T4), the mixture of trypsin inhibitors
T2/T3 and T4, and the chymotrypsin inhibitor C1 against the
proteases in the gut of various members of the Lepidoptera,
Coleoptera, Orthoptera and Diptera. In most cases, the pooled
peptides and the trypsin inhibitors had an equivalent effect
against the gut proteases with the degree of inhibition ranging
from 37-79% depending on the insect tested. The inhibitors had
negligible effect on the gut proteases of the potato tuber moth, P.
opercullela. The chymotrypsin inhibitor C1 also affected the
activity of the proteases but was less effective than the trypsin
inhibitors in five cases (W. cervinata, L. serricala, C.
zealandica, P. octo, sugar cane grub).
[0110] The experimental details are described in the legend to FIG.
14. The N. alata PI was more effective than Soybean Bowman-Birk
inhibitor in reducing cricket weight. It has shown that there is a
good correlation between the ability of a proteinase inhibitor to
inhibit the enzymes of the insect midgut and its effectiveness in
retarding the growth of insects in insect feeding trials
(Chisteller et al., 1992). FIG. 14 shows that the pooled PIs that
inhibited the gut proteases of the black field cricket (T.
commodus) by 70% in the in vitro assay retarded the growth of the
crickets by 30% in a feeding trial conducted over a 10 week period.
The correlation between in vitro assays and feeding trials has been
confirmed recently by Johnston and collegues (1993) working on
growth and development of Helicoverpa armigera. TABLE-US-00005
TABLE 2 HPLC retention time molecular peak (min) weight assigned
peptide* 1 15.5 5731.5 C1 5644.4 C1 minus Ser.sub.53 5616.4 C1
minus Asp.sub.1 & Ser.sub.53 55.29.3 C1 minus Asp.sub.1 2 20.5
5700.5 T2/T3 5728.5 T4 5585.4 T2/T3 minus Asp.sub.1 5613.5 T4 minus
Asp.sub.1 3 22.5 5725.5 T1 5610.5 T1 minus Asp.sub.1 4 24 5654.4 T1
minus Ala.sub.53 5641.4 T4 minus Ser.sub.53 5613.4 T2/T3 minus
Ser.sub.53 5539.4 T1 minus Asp.sub.1 & Ala.sub.53 5498.4 T2/T3
minus Asp.sub.1 & Ser.sub.53 5526.4 T4 minus Asp.sub.1 &
Ser.sub.53 *See FIG. 9 for designation of C1 and T1-T4.
[0111] TABLE-US-00006 TABLE 3 Effect of Nicotiona alata proteinase
inhibitors and Potato inhibitor II on casein hydrolysis by crude
gut extracts casein hydrolysis (% control) T2/T3, Insect NaPI C1 T4
H. armigera 33.2 32.7 30.3 H. punctigera 26.6 29.3 28.5 T. commodus
28.4 35.0 33.1 A. infusa 37.5 40.2 43.3 sugar cane 25.8 43.9 25.1
grub W. cervinata 22.9 82.9 20.4 E. postvitiana 39.7 45.4 41.2 S.
litura 28.1 33.6 24.8 P. opercullela 95.8 100 98.5 C. rufifacies
29.1 37.8 28.9 L. serricata 59.2 100 63.0 C. zealandica 31.7 54.7
32.0 P. octo 57.1 67.2 57.4 C. obliquana 51.1 49.1 45.5 A.
tasmaniae 28.3 34.2 39.5 Legend to Table 3 NaPI = N. alata
proteinase inhibitors pooled C1 = N. alata chymotrypsin inhibitor
(peak 1 from HPLC) T2/T3, T4 = N. alata trypsin inhibitors (peak 2
from HPLC) Heliothis armigera, Helicoverpa armigera, Tobacco
budworm, Lepidoptera Heliothis punctigera, Helicoverpa punctigera
Native budworm, Lepidoptera Teleogryllus commodus Black field
cricket, Orthoptera Agrotis infusa Common cutworm, adults known as
the Bogong moth, Lepidoptera Wiseana cervinata Porina, native to
New Zealand, Lepidoptera Lucilla sericata Green blow fly, Diptera,
assayed at pH 8 Chrysomya rufifacies Hairy maggot blov. fly,
Diptera, assayed at pH 8 Aphodius tasmaniae Tasmanian grass grub =
Black-headed pasture cockchafer, Coleoptera Costelytra zealandica
New Zealand grass grub, Coleoptera Spodoptera litura Tropical
armyworm, Lepidoptera Phthorimaea opercullela Potato tuber moth,
Lepidoptera Epiphyas postvittana Lightbrown apple moth
(leafroller), Lepidoptera Planotortrix octo Greenheaded leafroller,
Lepidoptera Ctenopseustis obliquana Brownheaded leafroller,
Lepidoptera Sugar cane grub
[0112] Those skilled in the art will appreciate that the invention
described herein is susceptible to variations and modifications
other than those specifically described. It is to be understood
that the invention includes all such variations and modifications.
The invention also includes all of the steps, features,
compositions and compounds referred to or indicated in this
specification, individually or collectively, and any and all
combinations of any two or more of said steps or features.
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Sequence CWU 1
1
19 1 1104 DNA Nicotiana alata 1 aaggcttgta ccttaaactg tgatccaaga
attgcctatg gagtttgccc gcgttcagaa 60 gaaaagaaga atgatcggat
atgcaccaac tgttgcgcag gcacgaaggg ttgtaagtac 120 ttcagtgatg
atggaacttt tgtttgtgaa ggagagtctg atcctagaaa tccaaaggct 180
tgtaccttaa actgtgatcc aagaattgcc tatggagttt gcccgcgttc agaagaaaag
240 aagaatgatc ggatatgcac caactgttgc gcaggcacga agggttgtaa
gtacttcagt 300 gatgatggaa cttttgtttg tgaaggagag tctgatccta
gaaatccaaa ggcttgtcct 360 cggaattgcg atccaagaat tgcctatggg
atttgcccac ttgcagaaga aaagaagaat 420 gatcggatat gcaccaactg
ttgcgcaggc aaaaagggtt gtaagtactt tagtgatgat 480 ggaacttttg
tttgtgaagg agagtctgat cctaaaaatc caaaggcctg tcctcggaat 540
tgtgatggaa gaattgccta tgggatttgc ccactttcag aagaaaagaa gaatgatcgg
600 atatgcacca actgctgcgc aggcaaaaag ggttgtaagt actttagtga
tgatggaact 660 tttgtttgtg aaggagagtc tgatcctaaa aatccaaagg
cttgtcctcg gaattgtgat 720 ggaagaattg cctatgggat ttgcccactt
tcagaagaaa agaagaatga tcggatatgc 780 acaaactgtt gcgcaggcaa
aaagggctgt aagtacttta gtgatgatgg aacttttgtt 840 ggtgaaggag
agtctgatcc tagaaatcca aaggcctgtc ctcggaattg tgatggaaga 900
attgcctatg gaatttgccc actttcagaa gaaaagaaga atgatcggat atgcaccaat
960 ggttgcgcag gcaagaaggg ctgtaagtac tttagtgatg atggaacttt
tatttgtgaa 1020 ggagaatctg aatatgccag caaagtggat gaatatgttg
gtgaagtgga gaatgatctc 1080 cagaagtcta aggttgctgt ttcc 1104 2 1360
DNA Nicotiana alata CDS (97)..(1200) 2 cgagtaagta tggctgttca
cagagttagt ttccttgctc tcctcctctt atttggaatg 60 tctctgcttg
taagcaatgt ggaacatgca gatgcc aag gct tgt acc tta aac 114 Lys Ala
Cys Thr Leu Asn 1 5 tgt gat cca aga att gcc tat gga gtt tgc ccg cgt
tca gaa gaa aag 162 Cys Asp Pro Arg Ile Ala Tyr Gly Val Cys Pro Arg
Ser Glu Glu Lys 10 15 20 aag aat gat cgg ata tgc acc aac tgt tgc
gca ggc acg aag ggt tgt 210 Lys Asn Asp Arg Ile Cys Thr Asn Cys Cys
Ala Gly Thr Lys Gly Cys 25 30 35 aag tac ttc agt gat gat gga act
ttt gtt tgt gaa gga gag tct gat 258 Lys Tyr Phe Ser Asp Asp Gly Thr
Phe Val Cys Glu Gly Glu Ser Asp 40 45 50 cct aga aat cca aag gct
tgt acc tta aac tgt gat cca aga att gcc 306 Pro Arg Asn Pro Lys Ala
Cys Thr Leu Asn Cys Asp Pro Arg Ile Ala 55 60 65 70 tat gga gtt tgc
ccg cgt tca gaa gaa aag aag aat gat cgg ata tgc 354 Tyr Gly Val Cys
Pro Arg Ser Glu Glu Lys Lys Asn Asp Arg Ile Cys 75 80 85 acc aac
tgt tgc gca ggc acg aag ggt tgt aag tac ttc agt gat gat 402 Thr Asn
Cys Cys Ala Gly Thr Lys Gly Cys Lys Tyr Phe Ser Asp Asp 90 95 100
gga act ttt gtt tgt gaa gga gag tct gat cct aga aat cca aag gct 450
Gly Thr Phe Val Cys Glu Gly Glu Ser Asp Pro Arg Asn Pro Lys Ala 105
110 115 tgt cct cgg aat tgc gat cca aga att gcc tat ggg att tgc cca
ctt 498 Cys Pro Arg Asn Cys Asp Pro Arg Ile Ala Tyr Gly Ile Cys Pro
Leu 120 125 130 gca gaa gaa aag aag aat gat cgg ata tgc acc aac tgt
tgc gca ggc 546 Ala Glu Glu Lys Lys Asn Asp Arg Ile Cys Thr Asn Cys
Cys Ala Gly 135 140 145 150 aaa aag ggt tgt aag tac ttt agt gat gat
gga act ttt gtt tgt gaa 594 Lys Lys Gly Cys Lys Tyr Phe Ser Asp Asp
Gly Thr Phe Val Cys Glu 155 160 165 gga gag tct gat cct aaa aat cca
aag gcc tgt cct cgg aat tgt gat 642 Gly Glu Ser Asp Pro Lys Asn Pro
Lys Ala Cys Pro Arg Asn Cys Asp 170 175 180 gga aga att gcc tat ggg
att tgc cca ctt tca gaa gaa aag aag aat 690 Gly Arg Ile Ala Tyr Gly
Ile Cys Pro Leu Ser Glu Glu Lys Lys Asn 185 190 195 gat cgg ata tgc
acc aac tgc tgc gca ggc aaa aag ggt tgt aag tac 738 Asp Arg Ile Cys
Thr Asn Cys Cys Ala Gly Lys Lys Gly Cys Lys Tyr 200 205 210 ttt agt
gat gat gga act ttt gtt tgt gaa gga gag tct gat cct aaa 786 Phe Ser
Asp Asp Gly Thr Phe Val Cys Glu Gly Glu Ser Asp Pro Lys 215 220 225
230 aat cca aag gct tgt cct cgg aat tgt gat gga aga att gcc tat ggg
834 Asn Pro Lys Ala Cys Pro Arg Asn Cys Asp Gly Arg Ile Ala Tyr Gly
235 240 245 att tgc cca ctt tca gaa gaa aag aag aat gat cgg ata tgc
aca aac 882 Ile Cys Pro Leu Ser Glu Glu Lys Lys Asn Asp Arg Ile Cys
Thr Asn 250 255 260 tgt tgc gca ggc aaa aag ggc tgt aag tac ttt agt
gat gat gga act 930 Cys Cys Ala Gly Lys Lys Gly Cys Lys Tyr Phe Ser
Asp Asp Gly Thr 265 270 275 ttt gtt tgt gaa gga gag tct gat cct aga
aat cca aag gcc tgt cct 978 Phe Val Cys Glu Gly Glu Ser Asp Pro Arg
Asn Pro Lys Ala Cys Pro 280 285 290 cgg aat tgt gat gga aga att gcc
tat gga att tgc cca ctt tca gaa 1026 Arg Asn Cys Asp Gly Arg Ile
Ala Tyr Gly Ile Cys Pro Leu Ser Glu 295 300 305 310 gaa aag aag aat
gat cgg ata tgc acc aat tgt tgc gca ggc aag aag 1074 Glu Lys Lys
Asn Asp Arg Ile Cys Thr Asn Cys Cys Ala Gly Lys Lys 315 320 325 ggc
tgt aag tac ttt agt gat gat gga act ttt att tgt gaa gga gaa 1122
Gly Cys Lys Tyr Phe Ser Asp Asp Gly Thr Phe Ile Cys Glu Gly Glu 330
335 340 tct gaa tat gcc agc aaa gtg gat gaa tat gtt ggt gaa gtg gag
aat 1170 Ser Glu Tyr Ala Ser Lys Val Asp Glu Tyr Val Gly Glu Val
Glu Asn 345 350 355 gat ctc cag aag tct aag gtt gct gtt tcc
taagtcctaa ctaataatat 1220 Asp Leu Gln Lys Ser Lys Val Ala Val Ser
360 365 gtagtctatg tatgaaacaa aggcatgcca atatgctctg tcttgcctgt
aatctgtaat 1280 atggtagtgg agcttttcca ctgcctgttt aataagaaat
ggagcactag tttgttttag 1340 ttaaaaaaaa aaaaaaaaaa 1360 3 368 PRT
Nicotiana alata 3 Lys Ala Cys Thr Leu Asn Cys Asp Pro Arg Ile Ala
Tyr Gly Val Cys 1 5 10 15 Pro Arg Ser Glu Glu Lys Lys Asn Asp Arg
Ile Cys Thr Asn Cys Cys 20 25 30 Ala Gly Thr Lys Gly Cys Lys Tyr
Phe Ser Asp Asp Gly Thr Phe Val 35 40 45 Cys Glu Gly Glu Ser Asp
Pro Arg Asn Pro Lys Ala Cys Thr Leu Asn 50 55 60 Cys Asp Pro Arg
Ile Ala Tyr Gly Val Cys Pro Arg Ser Glu Glu Lys 65 70 75 80 Lys Asn
Asp Arg Ile Cys Thr Asn Cys Cys Ala Gly Thr Lys Gly Cys 85 90 95
Lys Tyr Phe Ser Asp Asp Gly Thr Phe Val Cys Glu Gly Glu Ser Asp 100
105 110 Pro Arg Asn Pro Lys Ala Cys Pro Arg Asn Cys Asp Pro Arg Ile
Ala 115 120 125 Tyr Gly Ile Cys Pro Leu Ala Glu Glu Lys Lys Asn Asp
Arg Ile Cys 130 135 140 Thr Asn Cys Cys Ala Gly Lys Lys Gly Cys Lys
Tyr Phe Ser Asp Asp 145 150 155 160 Gly Thr Phe Val Cys Glu Gly Glu
Ser Asp Pro Lys Asn Pro Lys Ala 165 170 175 Cys Pro Arg Asn Cys Asp
Gly Arg Ile Ala Tyr Gly Ile Cys Pro Leu 180 185 190 Ser Glu Glu Lys
Lys Asn Asp Arg Ile Cys Thr Asn Cys Cys Ala Gly 195 200 205 Lys Lys
Gly Cys Lys Tyr Phe Ser Asp Asp Gly Thr Phe Val Cys Glu 210 215 220
Gly Glu Ser Asp Pro Lys Asn Pro Lys Ala Cys Pro Arg Asn Cys Asp 225
230 235 240 Gly Arg Ile Ala Tyr Gly Ile Cys Pro Leu Ser Glu Glu Lys
Lys Asn 245 250 255 Asp Arg Ile Cys Thr Asn Cys Cys Ala Gly Lys Lys
Gly Cys Lys Tyr 260 265 270 Phe Ser Asp Asp Gly Thr Phe Val Cys Glu
Gly Glu Ser Asp Pro Arg 275 280 285 Asn Pro Lys Ala Cys Pro Arg Asn
Cys Asp Gly Arg Ile Ala Tyr Gly 290 295 300 Ile Cys Pro Leu Ser Glu
Glu Lys Lys Asn Asp Arg Ile Cys Thr Asn 305 310 315 320 Cys Cys Ala
Gly Lys Lys Gly Cys Lys Tyr Phe Ser Asp Asp Gly Thr 325 330 335 Phe
Ile Cys Glu Gly Glu Ser Glu Tyr Ala Ser Lys Val Asp Glu Tyr 340 345
350 Val Gly Glu Val Glu Asn Asp Leu Gln Lys Ser Lys Val Ala Val Ser
355 360 365 4 24 PRT Nicotiana alata 4 Lys Ala Cys Thr Leu Asn Cys
Asp Pro Arg Ile Ala Tyr Gly Val Cys 1 5 10 15 Pro Arg Ser Glu Glu
Lys Lys Asn 20 5 58 PRT Nicotiana alata 5 Asp Arg Ile Cys Thr Asn
Cys Cys Ala Gly Thr Lys Gly Cys Lys Tyr 1 5 10 15 Phe Ser Asp Asp
Gly Thr Phe Val Cys Glu Gly Glu Ser Asp Pro Arg 20 25 30 Asn Pro
Lys Ala Cys Thr Leu Asn Cys Asp Pro Arg Ile Ala Tyr Gly 35 40 45
Val Cys Pro Arg Ser Glu Glu Lys Lys Asn 50 55 6 58 PRT Nicotiana
alata 6 Asp Arg Ile Cys Thr Asn Cys Cys Ala Gly Thr Lys Gly Cys Lys
Tyr 1 5 10 15 Phe Ser Asp Asp Gly Thr Phe Val Cys Glu Gly Glu Ser
Asp Pro Arg 20 25 30 Asn Pro Lys Ala Cys Pro Arg Asn Cys Asp Pro
Arg Ile Ala Tyr Gly 35 40 45 Ile Cys Pro Leu Ala Glu Glu Lys Lys
Asn 50 55 7 58 PRT Nicotiana alata 7 Asp Arg Ile Cys Thr Asn Cys
Cys Ala Gly Lys Lys Gly Cys Lys Tyr 1 5 10 15 Phe Ser Asp Asp Gly
Thr Phe Val Cys Glu Gly Glu Ser Asp Pro Lys 20 25 30 Asn Pro Lys
Ala Cys Pro Arg Asn Cys Asp Gly Arg Ile Ala Tyr Gly 35 40 45 Ile
Cys Pro Leu Ser Glu Glu Lys Lys Asn 50 55 8 58 PRT Nicotiana alata
8 Asp Arg Ile Cys Thr Asn Cys Cys Ala Gly Lys Lys Gly Cys Lys Tyr 1
5 10 15 Phe Ser Asp Asp Gly Thr Phe Val Cys Glu Gly Glu Ser Asp Pro
Lys 20 25 30 Asn Pro Lys Ala Cys Pro Arg Asn Cys Asp Gly Arg Ile
Ala Tyr Gly 35 40 45 Ile Cys Pro Leu Ser Glu Glu Lys Lys Asn 50 55
9 58 PRT Nicotiana alata 9 Asp Arg Ile Cys Thr Asn Cys Cys Ala Gly
Lys Lys Gly Cys Lys Tyr 1 5 10 15 Phe Ser Asp Asp Gly Thr Phe Val
Cys Glu Gly Glu Ser Asp Pro Arg 20 25 30 Asn Pro Lys Ala Cys Pro
Arg Asn Cys Pro Gly Arg Ile Ala Tyr Gly 35 40 45 Ile Cys Pro Leu
Ser Glu Glu Lys Lys Asn 50 55 10 54 PRT Nicotiana alata 10 Asp Arg
Ile Cys Thr Asn Cys Cys Ala Gly Lys Lys Gly Cys Lys Tyr 1 5 10 15
Phe Ser Asp Asp Gly Thr Phe Ile Cys Glu Gly Glu Ser Glu Thr Ala 20
25 30 Ser Lys Val Asp Glu Tyr Val Gly Glu Val Glu Asn Asp Leu Gln
Lys 35 40 45 Ser Lys Val Ala Val Ser 50 11 13 PRT Nicotiana alata
11 Asp Arg Ile Cys Thr Asn Cys Cys Ala Gly Thr Lys Gly 1 5 10 12 13
PRT Nicotiana alata 12 Asp Arg Ile Cys Thr Asn Cys Cys Ala Gly Lys
Lys Gly 1 5 10 13 6 PRT Nicotiana alata 13 Lys Ala Cys Thr Leu Asn
1 5 14 5 PRT Nicotiana alata 14 Glu Glu Lys Lys Asn 1 5 15 53 PRT
Artificial Sequence synthetic peptide 15 Asp Arg Ile Cys Thr Asn
Cys Cys Ala Gly Thr Lys Gly Cys Lys Tyr 1 5 10 15 Phe Ser Asp Asp
Gly Thr Phe Val Cys Glu Gly Glu Ser Asp Pro Arg 20 25 30 Asn Pro
Lys Ala Cys Thr Leu Asn Cys Asp Pro Arg Ile Ala Tyr Gly 35 40 45
Val Cys Pro Arg Ser 50 16 53 PRT Artificial Sequence synthetic
peptide 16 Asp Arg Ile Cys Thr Asn Cys Cys Ala Gly Thr Lys Gly Cys
Lys Tyr 1 5 10 15 Phe Ser Asp Asp Gly Thr Phe Val Cys Glu Gly Glu
Ser Asp Pro Arg 20 25 30 Asn Pro Lys Ala Cys Pro Arg Asn Cys Asp
Pro Arg Ile Ala Tyr Gly 35 40 45 Ile Cys Pro Leu Ala 50 17 53 PRT
Artificial Sequence synthetic peptide 17 Asp Arg Ile Cys Thr Asn
Cys Cys Ala Gly Lys Lys Gly Cys Lys Tyr 1 5 10 15 Phe Ser Asp Asp
Gly Thr Phe Val Cys Glu Gly Glu Ser Asp Pro Lys 20 25 30 Asn Pro
Lys Ala Cys Pro Arg Asn Cys Asp Gly Arg Ile Ala Tyr Gly 35 40 45
Ile Cys Pro Leu Ser 50 18 53 PRT Artificial Sequence synthetic
peptide 18 Asp Arg Ile Cys Thr Asn Cys Cys Ala Gly Lys Lys Gly Cys
Lys Tyr 1 5 10 15 Phe Ser Asp Asp Gly Thr Phe Val Cys Glu Gly Glu
Ser Asp Pro Lys 20 25 30 Asn Pro Lys Ala Cys Pro Arg Asn Cys Asp
Gly Arg Ile Ala Tyr Gly 35 40 45 Ile Cys Pro Leu Ser 50 19 53 PRT
Artificial Sequence synthetic peptide 19 Asp Arg Ile Cys Thr Asn
Cys Cys Ala Gly Lys Lys Gly Cys Lys Tyr 1 5 10 15 Phe Ser Asp Asp
Gly Thr Phe Val Cys Glu Gly Glu Ser Asp Pro Arg 20 25 30 Asn Pro
Lys Ala Cys Pro Arg Asn Cys Asp Gly Arg Ile Ala Tyr Gly 35 40 45
Ile Cys Pro Leu Ser 50
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